Methods and compositions for diagnosing autoimmune insulin dependant diabetes mellitus

ABSTRACT

Disclosed are compositions and methods for diagnosing autoimmune insulin dependent diabetes mellitus. In general, diagnostic methods of the invention include testing for the presence of an autoimmune immunoglobulin in a suspected patient&#39;s serum wherein the immunoglobulin is identified by its ability to interfere with the glucose transporting activity of a pancreatic islet cell glucose transporter. The inventors have discovered that the presence of such an autoimmune antibody in patient&#39;s serum is diagnostic of autoimmune insulin dependant diabetes mellitus. In particular aspects the diagnostic assay of the invention involves the incubation of isolated and dispersed islet cells, such as rat islet cells, in the presence of immunoglobulin obtained from the patient. Following such an incubation, the islet cells are tested for their ability to uptake glucose. A diagnosis is made through a determination that immunoglobulins in the serum of the patient specifically inhibit the uptake of glucose by the islet cells.

The Government may own certain rights in this invention pursuant to NIHGrant AM-02700.

This application is a continuation of U.S. Ser. No. 483,224, filed Feb.20, 1990, now U.S. Pat. No. 5,175,085.

BACKGROUND

1. Field of the Invention

The present invention relates to compositions and methods for diagnosingautoimmune insulin dependent diabetes mellitus (IDDM). In particular,the invention concerns the diagnosis of IDDM through the detection of aparticular autoimmune immunoglobulin (Ig) in patient sera, an Ig whichinterferes with a pancreatic islet cell-localized glucose transporter.

2. Description of the Related Art

Insulin Dependent Diabetes Mellitus ("Type I") represents 20% of allhuman diabetes, and is the most serious form of the disease, with thehighest morbidity and mortality. Progression of the disease isassociated with a major loss of pancreatic islet β cell function andcell destruction (1). This loss of pancreatic β cells which isapparently quite cell-specific in its early stages since other cellpopulations which comprise the islets of Langerhans (α cells, δ cells)are unaffected (2).

The series of autoimmunologic events which give rise to IDDM, or whichare otherwise involved with IDDM onset, are poorly understood. However,the disease involves a progressive reduction in the function ofpancreatic islets of Langerhans β cells. It has, for example, beenreported that a preferential loss of insulin response to glucose occurswithout a corresponding reduction of the response to certain non-glucosesecretagogues, such as amino acids or isoproterenol, during developmentof IDDM (3).

Currently available diagnostic procedures such as blood and urinedeterminations diagnose IDDM only after the onset of symptomatologyassociated with the disease, when β cell destruction is almost complete.This reduces or eliminates an ability to initiate intervention early on,when it is theoretically possible to arrest the destructive process. Forthis reason, a simple, rapid, and inexpensive diagnostic test capable ofcorrectly identifying a IDDM patients prior to the onset of clinicaldisease is needed. Currently, islet cell antibodies and insulinautoantibodies identify only about 60% of such patients.

An autoimmune etiology has been implicated in a large percentage of IDDMcases, which have led researchers to try to identify a particular targetor antigen. For example, Kanatsuna et al. have reported that plasmaobtained from IDDM patients soon after onset will inhibitglucose-stimulated insulin secretion by rat islets (4). Furthermore, theresults from several studies employing various techniques, includingimmunofluorescence on tissue section, chromium⁵¹ -release, etc., havedemonstrated that antibodies reactive with pancreatic islet cells areoften present at the time of diagnosis of IDDM (1). Other studies haveidentified antibodies to a 64 kd islet membrane protein, said to bedetectable prior to the onset of clinical disease, in 73% of new onsetIDDM patients (6-8). However, no biological function has been ascribedthis antigen, and it has not as yet been isolated or furthercharacterized.

For the foregoing and other reasons, a need exists for a diagnostic testfor IDDM that addresses one or more of the problems associated withpreviously available tests. For example, a test that can predict thepotential for IDDM development prior to its onset, thus allowing timefor intervention therapy would prove particularly useful, as would atest capable of faithfully identifying pre-onset IDDM patients with ahigh sensitivity. It is believed that the present invention, directed tomethods for the detection of particular autoimmune antibodies, addressesat least some of these or other disadvantages associated with previousapproaches to IDDM diagnosis.

SUMMARY OF THE INVENTION

The present invention is directed in general to the diagnosis ofautoimmune Type I diabetes (IDDM) through the detection of a particularclass of autoimmune immunoglobulins--those having immunospecificity fora pancreatic islet β-cell glucose transporter, or for a separate,functionally associated protein. The islet β-cell glucose transporter orassociated protein, while apparently similar in function to othertransporters, is immunologically distinct from those found in skeletalmuscle, red blood, adipocyte-containing tissue, and even brain tissue.The invention evolves out of the inventors discovery that suchantibodies are associated with the onset of IDDM and can be used asearly diagnostic indicators of the disease. The invention has thesurprising and unique feature of detecting, in a single assay,immunological and functional abnormalities. This is so sinceneutralization of the antigen, the glucose transporter, by the antibodymay cause the functional defect in insulin secretion.

During their development of the invention, the inventors proposed thatan early event in the onset of IDDM is the appearance of anti-β cellimmunoglobulins which react with β-cell- and liver cell-localizedglucose transporter or functionally associated protein. Thisanti-glucose transporter is believed to bind to the transporter andthereby sterically hinder or otherwise interfere with the uptake ofglucose by β-cells. The resultant lack of glucose greatly compromisesthe β-cell and ultimately leads to a selective loss of β-cell function,as well as β-cells themselves.

The invention is thus directed in part to a method for diagnosingautoimmune insulin dependent diabetes mellitus (IDDM) which includestesting the sera of a suspected diabetic or candidate for early onsetdiabetes, for the presence of an autoimmune immunoglobulin identified byits ability to interfere with the glucose transporting activity ofpancreatic islet cells. The inventors have discovered that the presenceof such an antibody in a patient's serum is diagnostic of Type Idiabetes, and it is believed to be an early diagnostic indicator ofsubsequent development of diabetes.

As used herein, the phrase "islet cell glucose transporter" refers toone or more of the protein or proteins which take part in thefacilitated transport of glucose into islet β-cells. The term is alsointended to include associated structures--perhaps sterically associatedin terms of a proximal location, for example, on the β-cell surface--solong as the structure comprises antigenic epitopes which, whenrecognized and bound by autoimmune antibodies, results in thesuppression of glucose uptake. Such epitopes may be natural antigenicepitopes of the glucose transporter. Alternatively, such epitopes may besynthetically produced to mimic antigenic regions of the glucosetransporter, and, in any case, will stimulate production of antigencapable of interrupting transport of glucose into islet β-cells.

In certain embodiments, testing for the presence of an anti-β-cellglucose transporter include steps of 1) preparing an admixture of theislet cell glucose transporter together with immunoglobulin (Ig) fromthe patient; 2) incubating the admixture under conditions appropriatefor the formation of immunocomplexes; and 3) testing for the formationof immunocomplexes between the islet cell glucose transporter and thepatient's Ig, indicative of the presence of the anti-β cell glucosetransporter Ig in the patient's sera.

In particular aspects, testing for the anti-transporter autoimmuneimmunoglobulin includes testing for a relative inhibition of the rate ofglucose uptake by the islet cells, with such an inhibition indicatingthe presence of the anti-transporter antibody. This embodiment reliesupon an ability to assay for glucose transporter biologic activity. Thisarises out of the inventors observation that the rate of glucose uptake,particularly the initial rate, is relatively inhibited in the presenceof the anti-transporter immunoglobulin. The term "relatively inhibited"is intended to refer broadly to inhibition "relative" to some control,such as the inhibition of the islet cell antigen as compared tonon-islet cell, by techniques such as comparison of the patient'simmunoglobulin to a control immunoglobulin preparation, or by comparisonof the uptake of glucose in relation to a control metabolite or thelike.

In certain aspects, a biological assay is employed to test for thepresence of antibodies having anti-glucose transporter activity, e.g.,plus and minus incubation with patient's immunoglobulins. In theseinstances, one will desire to obtain the transporter in a biologicallyactive form. The inventors have found that the most convenient means isto simply employ isolated rat islet cells, obtained, e.g., followingcollagenase digestion of rat pancreas and Ficoll gradient purification.Isolated rat islet cells can be readily used to measure glucose uptakein connection with the practice of the present invention in that the ratβ-cell transporter is apparently immunocrossreactive with the humantransporter. Thus, the rat islet cell transporter is recognized, andinhibited, by human anti-β-cell glucose transporter immunoglobulins.Other embodiments of biologically active glucose transporter may beemployed in place of rat islet cells, including glucose transporterexpressed in non-β-cells or synthetically (9).

It is preferable to prepare immunoglobulin G (IgG) from the patient'ssera prior to testing for the antibody. This is due to possibleinterfering substances which may be present in the patient's sera or Igand can give a high background or reduced sensitivity or selectivity tothe assay. Using purified IgG is particularly desirable where a bioassayis to be performed. In that the autoimmune Ig identified in connectionwith the present invention will generally be IgG, the use of the IgGfraction in assays hereof is to be encouraged. IgG of suitable purityhas been routinely obtained by the inventors by purification of sera ona protein A Sepharose column (10). Of course, other suitable methods forpreparing IgG are known and these could be successfully employed,including polyetheneglycol precipitation and ammonium sulfatefractionation (4).

Glucose uptake may be concomitantly measured through the use of areadily identifiable glucose analog that will function as a substratefor the transporter. Exemplary analogs, particularly useful inconnection with the present invention, are those which can incorporatedetectable ligands, radioactive groups, H³, C¹⁴ and the like. Apreferred analog is 3-0-methyl-β-D-glucose which is both easy to prepareand into which such detectable ligands can readily be introduced. While3-0-methylated analogs are preferred, other analogs or substrates couldbe employed if desired, such as 2-deoxy-glucose.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Time course of the uptake of 3-0-Methyl-β-D-Glucose (opencircles) and L-Glucose (closed circles) by Dispersed Rat Islet Cells.

Individual points on each curve represent the mean±SEM from duplicatedeterminations made in six independent experiments. Uptake measurementswere performed on dispersed cells from 250 islets for each time point.

FIGS. 2A and 2B. Effects of purified IgG from normal humans (opencircles) and new-onset IDDM patients (closed circles) on the uptake of3-0-methyl-β-D-glucose and L-leucine by dispersed rat islet cells.

FIG. 2A. Uptake of 3MG by islet cells after treatment with IgG fromnormal humans (n=28) and new-onset IDDM patients (n=27).

FIG. 2B. Uptake of L-leucine by islets after treatment with IgG fromnormal humans (n=15) and new-onset IDDM patients (n=9).

Uptake assays were performed using coded IgG samples. Samples weredecoded and data from duplicate determinations at each time point foreach individual IgG sample were segregated into diagnosis groups andused to determine the mean±SEM of uptake at each time point shown in thecurves. L-leucine uptake was measured on the same islet preparation as3MG uptake for the indicated number of IgG samples. The statisticaldifferences in the time points of the curves is indicated by an (*)where p<0.01.

FIG. 3. Distribution of the initial rates of 3-0-methyl-β-D-glucoseuptake by rat islet cells in the presence of IgG from each individualafter segregation in to diagnosis groups.

Initial rates of uptake of 3MG uptake were determined as described inthe Methods sections for each IgG sample from 28 normal humans, 27 IDDMpatients, and 5 NIDDM patients. The horizontal solid line is the mean3MG uptake in the presence of IgG from each group and the dashed linerepresents one standard deviation from the mean in each group.

FIG. 4. Effects of IgG from Normal Subjects (open circles) and New-OnsetPatients (closed circles) on the Glucose Concentration Dependence ofGlucose Uptake by Rat Islet Cells.

The initial rates of 3-0-methyl-β-D-glucose uptake were determined asdescribed in Methods. The islet cells were incubated with IgGs from 3normal subjects (open circles) and 3 IDDM patients (closed circles) and3-0-methyl-β-D-glucose uptake was measured at 60, 30, 15, 5, 2, and 1 mM3-0-methyl-β-D-glucose concentrations. The results are presented as theEadie-Hofstee transformation of the mean initial velocity measurements.The solid lines represent the results obtained with islet cells assayedin buffer alone.

FIGS. 5A and 5B. Effects of IgG fractions from normal subjects (opencircles) and IDDM patients (closed circles) on the uptake of3-0-methyl-β-D-glucose by human erythrocytes (FIG. 5A) and rathepatocytes (FIG. 5B).

Uptake of 3MG was measured as described for islet cell uptake using IgGpreparations from normal subjects and 3 IDDM patients. The results shownare the mean±SEM values for each time point.

FIG. 6. Effect of preincubation of IgG from IDDM patients with cells orcell membranes from islets, liver, erythrocytes, or kidney brush borderon 3-methyl glucose uptake by islet cells. The preparation of themembranes and their incubation with the IgG fractions are described indetail in the Methods section. Ab is uptake in the presence of IgG notpreincubated with any tissue preparation.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Autoimmunity and a progressive loss of β-cell function are features ofinsulin-dependent diabetes mellitus (IDDM). In the development of theinvention, characteristics such as these led the inventors to considerthat a part of the glucose recognition apparatus of β-cells was involvedin the autoimmune response. Contrary to many existing theories (4), itwas considered by the inventors that a target of the autoimmune responsewas a pancreatic β-cell-localized glucose transporter, recognized byautoimmune antibodies in connections with the onset of the disease. Aseries of studies have been conducted by the inventors which support aconclusion that the glucose transporter, or perhaps a physically orfunctionally associated antigenic structure, is indeed an autoimmunetarget in new onset IDDM.

In an exemplary set of studies, set forth herein, the uptake of3-0-methyl-β-D-glucose by dispersed rat islets of Langerhans wasmeasured after treatment with IgG obtained from 28 control humans, 27new-onset IDDM humans and 5 noninsulin-dependent diabetic (NIDDM)humans. In 26 of 27 (94%) IgG fractions from IDDM patients, initialrates of 3-0-methyl-β-D-glucose uptake were found to be inhibited belowone standard deviation from the mean of controls (p<0.001). This effectwas not a general permeability decrease as there was no effect onL-leucine uptake by islet cells after treatment with these IgGfractions. Further, the IgG fractions from IDDM patients did not inhibituptake of 3-0-methyl-β-D-glucose by human erythrocytes or rathepatocytes. These studies demonstrated that IgG from humans withnew-onset IDDM inhibits glucose uptake by rat islet cells by recognizingthe glucose transporter of these cells or an unrelated protein which isinvolved structurally or functionally in islet cell glucose transport.

In more particular terms, the method of invention involves testing forthe presence of an autoimmune immunoglobulin in serum of a patientsuspected of having autoimmune IDDM, wherein the immunoglobulin isidentified by its ability to interfere with the glucose transportingactivity of pancreatic cell glucose transporter. The presence of such animmunoglobulin in the patient serum has been found by the inventors tobe diagnostic of autoimmune IDDM. Most conveniently, the assay isperformed by testing for the presence of immunoglobulins that arecapable of inhibiting glucose transport function in isolated isletcells. Preferably, islet cells are isolated from rat pancreata anddispersed to form an essentially single cell suspension. Ideally a cellsuspension is prepared wherein cell aggregation is minimized. In that ithas been found that maintenance of cell viability is important to thesuccessful practice of the preferred method of the invention, it may bedesirable to determine cell viability by a procedure such as fluoresceindiacetate uptake, or uptake of another vital dye which is indicative ofa functional cell membrane transport system and therefore viability.Typically one will desire to obtain at least about 70% of the cells asviable cells, and more preferably at least about 70% viable cells.

Following isolation and dispersion of islet cells, the cells areincubated in the presence of an antibody solution prepared from sera ofthe patient to be tested. Preferably, the dispersed cells are incubatedwith a immunoglobulin G (IgG) fraction prepared from the patient's sera.While more conventional methods of IgG preparation such as ammoniumsulfate fractionation can be employed to prepare IgG, a more preferredapproach involves the use of a protein A-Sepharose or like adsorbent.Protein A-Sepharose chromatography has been found to provide a simpleand expedient means for obtaining relatively purified IgG. While the useof IgG is preferred, it is believed by the inventors that totalimmunoglobulin or even serum may be used directly in the assay ifdesired. In any event, in initial incubations with isolated islet cellsone will typically desire to employ patient antibody-containingcompositions at a concentration wherein the IgG concentration in thefinal incubation admixture will be on the order of 5-15 mg/ml, andpreferably about 8-10 mg IgG/ml. This will represent approximately 10⁶islet cells per mg IgG.

In the measurement of glucose uptake by the dispersed cells, one maydesire to incubate the dispersed cells in an antibody solution togetherwith a labeled indicator or marker which will allow one to determineintracellular space. An example of such a marker of intracellular spaceis urea, which will penetrate the cells and occupy total intracellularspace in a non-facilitated fashion. By measuring the uptake of anintracellular space marker such as urea, one is able to determine thetotal amount of intracellular space into which the glucose to besubsequently measured is taken up. Such a measurement, while not crucialto the practice of the invention, allows one to determine with greaterprecision the measurement of glucose uptake. Other suitableintracellular space markers are well known in the art and include, forexample, urea or water, or the like.

Following incubation with an intracellular space marker, the cells arefurther incubated in the presence of the selected glucose analogue. Apreferred analogue is 3-0-methyl-β-D-glucose in that this analogue canbe readily prepared and is taken up by viable cells in a facilitatedfashion just as glucose itself is but is not metabolized by these cellswhile glucose is metabolized. In preparing the selected glucoseanalogue, one will generally desire to incorporate a ligand that isdistinct from the intracellular marker, where one is employed. Thereason for this is to allow one to separately measure the glucose uptakein relation to the intracellular space. Thus, for example, one maydesire to employ a C¹⁴ ligand in connection with an intracellular spacemarker such as urea and a H³ ligand in connection with the glucoseanalogue.

Glucose uptake may be initiated by admixing an antibody-incubated cellsuspension with the glucose analogues for a selected period of time. Itmay be desirable to terminate uptake by sedimenting the cells through ahydrophobic phase, such as a dibutyl/dinonyl phthalate phase, into acushion such as a glucose cushion by centrifugation. Such a procedure isdesirable in that it separates only viable cells for uptake measurementand minimizes extracellular space which can interfer with the precisionof the measurement.

Following termination of incubation, uptake determinations may be madethrough scintillation counting of the cells. The uptake of separateisotopes, corresponding to intracellular space and glucose uptake,respectively, thus may be determined by reference to the scintillationcounter window that is appropriate for the particular isotope. Whereboth C¹⁴ and tritium are employed, respectively, one will desire toobtain a counting of the incubated cell material in both a tritium and aC¹⁴ window of the counter.

In that one will generally desire to measure the initial rates ofglucose analogue uptake, uptake is measured at selected intervalsfollowing initiation of glucose uptake. It has been found that timepoints in the 3 to 15 second time frame, measured from an initiation ofglucose uptake, is most preferred. The determination of initial rates ofglucose uptake have been found by the present inventors to reflect themost reliable means of detecting the presence of the anti-transporterantibody in the patient's serum. Measurements taken at, e.g., timepoints such as 3, 6 and 15 seconds of incubation, can then be expressedin terms of a mean for each point.

A determination of the relative reduction in the initial rate of glucoseuptake can be performed by comparing the inhibition of glucose uptake byislet cells treated with IgG from suspect patients to glucose uptake byislet cells treated with IgG from known normal individuals. As a secondlevel of assessment, one can compare the inhibition of glucose uptakeand islet cells versus the uptake of a metabolite that is taken up byislet cells by transport other than by the glucose transporter. In suchan embodiment the uptake of glucose will be inhibited by theanti-transporter antibody whereas the uptake of the non glucosemetabolite will not. Suitable metabolites which can serve as uptakecontrols include amino acids such as L-leucine, L-arginine, orL-alanine. Other useful metabolites include ions, organic acids, or thelike.

Therefore, to determine the control uptake, whether it be a glucoseuptake by non-islet cells or a non-glucose metabolite uptake by isletcells, control assays may be run in parallel with assays measuring isletcell glucose uptake. The uptake determined for controls are thencompared to the uptake determined for islet cell glucose uptake. Fromthis comparison it is determined whether there is a relative inhibitionof glucose uptake in the presence of the suspected patient'simmunoglobulin.

For the purposes of the present invention, it is believed that arelative inhibition of on the order of 30 to 70%, will indicate thepresence of an anti-transporter immunoglobulin in the patient's serum.Generally, one will see an inhibition of on the order of 50% in suchpatients. However, it is believed that inhibitions of on the order of 30to 70% are useful indicators of the anti-transporter antibody in suchranges as submitted to be diagnostic of the disease.

The following example illustrates various preferred embodiments forcarrying out the invention. The studies set forth below were conductedin part through the application of standard laboratory practices of theinventors as well as procedures developed by the inventors or found towork well in the practice of the invention. Various modifications,rearrangements of steps substitutions and the like, will be apparent tothe skilled artisan in light of the examples which follow.

DIAGNOSIS OF AUTOIMMUNE IDDM USING DISPERSED RAT ISLET CELLS A.Procedures Employed

1. Patient Populations

Serum for IgG purification was obtained from 27 new onset IDDM patients(ages 6-34), 28 nondiabetic controls (ages 22-64) and 5 patients withNIDDM (ages 55-64). In the IDDM patients the serum was obtained within 2months of diagnosis; in 1 patient the diagnosis had been made 8 monthsearlier. Each serum was assigned a code number by an outside colleagueand all subsequent work was performed on coded specimens. The IgGfractions from all diabetic patients were tested for the presence ofcytoplasmic islet cell antibodies using the method of Krell & Rabin (9).

2. Immunoglobulin G (IqG) Purification

Protein A-Sepharose (Pharmacia Fine Chemicals, Uppsala, Sweden) wasprepared by extensive washing and pre-equilibration with 0.1 M sodiumphosphate, pH 7.0. Sera from patients were diluted with an equal volumeof 0.1 M sodium phosphate pH 7.0 and 4 ml of sample were mixed with 2 mlProtein A-Sepharose. Following 30 minute incubation with inversionmixing at room temperature, the suspension was transferred to a columnand washed with 20 ml of 0.1 M sodium phosphate, pH 7.0. IgG was elutedfrom the column using 10 ml of 0.5 M acetic acid in 0.89% saline pH 3.0.During elution of antibody from the column, the pH of the eluted wasmonitored and immediately adjusted to pH 7.0 with 3.3 M Trishydroxyamino methane (free base). The eluate was concentrated anddialyzed against phosphate-buffered saline. IgG concentrations used inthis study were 9.0±0.3 mg/ml, which represents approximately 10⁶ isletcells per mg IgG.

3. Islet Isolation and Dispersal

Islets for each experiment were isolated from the pancreata of 15-20male Wistar rats (200-250 g) using a modification of the method of Naberet al. (12). Briefly, pancreata were inflated with 20 ml of chilledHanks balanced salt solution (HBSS) containing 0.5% bovine serum albuminand the digested material was washed three times in HBSS containing 0.5%BSA. The final sediment was resuspended in 4 mls 25% Ficoll in HBSS andwas overlaid with 2 mls each of 23%, 20.5% and 11% Ficoll (w/w).Gradients were centrifuged at 700× g for 15 minutes at 4° C. and isletswere harvested from the 11%/20.5% interface with a plastic pipet. Ficollwas removed by washing the islets with HBSS containing 0.5% bovine serumalbumin by centrifugation at 700 kg for 2 minutes at room temperature.From 5000 to 8000 islets were routinely obtained from 20 rats.

Purified islets were resuspended in Ca²⁺, Mg²⁺ -free HBSS containing 3mM ethyleneglycol-bis-(β-amino ethyl ether) N, N'tetracetic acid (EGTA)and incubated at 37° C. for 15 minutes. During incubation, islets weresubjected to gentle aspiration into and out of a plastic pipet. Thisprocedure yielded a cell suspension which was mostly single cells with afew three to five cell aggregates. Fluorescein diacetate uptake revealedthat 70±5% of the cells were viable (13).

4. Uptake Measurement

Dispersed cells from 3500 islets were incubated in 0.7 ml antibodysolution containing a final concentration of 2 mM [¹⁴ C] urea (0.5μCi/μmole) as an intracellular space marker at 37° C. for 20 minutes.Cells were further incubated for 10 minutes at 15° C. before assay ofuptake. Uptake of glucose analogues was determined using a modificationof the method of Gorus et al. (14).

3-0-methyl-β-D-glucose was prepared as follows: 50 μl of 1 M glucosecontaining 10 mM EDTA and 0.1% sodium dodecylsulfate (SDS), pH 8.0 wasplaced in the bottom of 400 μl microfuge tubes. This solution wasoverlaid with 150 μl of a dibutyl phthalate/dinonyl phthalate (4:1)mixture. Following 30 seconds of centrifugation in a Beckman microfuge,50 μl of phosphate-buffered saline containing [¹⁴ C] urea (2 mM, 0.5μCi/μmole) and 20 mM 3-0-β-D [³ H] methyl-glucopyranoside (5 μCi/μmole)was layered over the dibutyl/dinonyl phthalate phase. The tubes werethen preincubated for 20 minutes at 15° C.

Uptake of the glucose analogue was initiated by adding 50 μl of the cellsuspension preincubated with urea to the 50 μl phosphate-buffered salinephase containing labeled glucose analogues and urea. Uptake wasterminated by starting the microfuge and sedimenting the cells throughthe dibutyl/dinonyl phthalate phase into the 1 M glucose, 10 mM EDTA,0.1% SDS. A 50 μl portion of the supernatant was removed from each tubefor specific activity calculations and a 35 μl portion of the 1 Mglucose, 10 mM EDTA, 0.1% SDS phase was removed for uptake determinationand counted in a Beckman LS 5801 liquid scintillation counter.

Uptake was measured in duplicate for the times indicated in eachexperiment. Data were reduced to dpm for each isotope using the residentcalculation program of the counter and were expressed as mmoles/literislet space. Corrections for extracellular space as judged by zero-timeL-glucose measurements were 19, 22 and 28 percent of the total ureaspace in experiments performed in buffer alone, buffer containing IgGfrom normal humans, and buffer containing IgG from IDDM patients,respectively. Initial rates of 3MG uptake were derived from the 3, 6,and 15 second time points. Measurement of L-[1, ³ H] glucose andL-[3,4,5,³ H]-leucine uptake were made using the same procedure. Dataare expressed as mean±standard error of the mean (SEM) for each timepoint.

5. Statistical Analyses

Data were decoded, segregated according to diagnosis and compared byusing an unpaired two-tailed t-test. The mean±SEM for all data arereported in cumulative time courses and initial rates of substrateuptake.

6. Adsorption of IgG Preparations

Adsorption studies were carried out to determine if the glucosetransport inhibitory activity of the IgG fractions was abolished bypreincubation of with various tissues that contain glucose transporterssimilar or dissimilar to β-cells. We prepared unsealed red blood cellghosts (13), liver plasma membranes (14), and kidney brush borderspecimens (15). The IgG preparations were diluted to 4 mg IgG/ml inphosphate-buffered saline and incubated with dispersed islet cells from40 rats (.sup.˜ 20,000 islets), 20 mb liver plasma membrane protein, 20mb rat red blood cell ghost protein, 20 mg kidney brush border membraneprotein or buffer alone for 3 hr at 4° C. The mixtures were thencentrifuged at 800× g for 5 minutes and twice at 50,000× g for 10 min toremove islet cells or membranes. The supernatants were carefully removedand stored at 4° C. overnight, after which they were added to dispersedrat islet cells. Following incubation the cells were assayed for 3MGuptake as described above.

B. Results of the Studies

1. Clinical Composition of Patient Populations

The sex. age. diagnosis, treatment and islet cell antibody status of allIDDM patients are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Characteristics of Diabetic Patients and Normal Subjects                      ______________________________________                                        Patent              Age     Islet Cell Antibody                                                            Type 1 Sex (years) (cytoplasmic)                 ______________________________________                                          1 M 19 +                                                                      2 M 7 +                                                                       3 M 13 +                                                                      4 M 10 +                                                                      5 F 8 +                                                                       6 F 7 +                                                                       7 F 11 +                                                                      8 F 8 +                                                                       9 F 7 +                                                                       10 F 8 +                                                                      11 M 12 +                                                                     12 M 14 +                                                                     13 F 24 +                                                                     14 M 6 +/-                                                                    15 M 11 +/-                                                                   16 F 7 -                                                                      17 F 11 -                                                                     18 F 11 -                                                                     19 F 13 -                                                                     20 M 9 -                                                                      21 F 12 -                                                                     22 M 19 -                                                                     23 F 8 -                                                                      24 F 12 -                                                                     25 M 6 -                                                                      26 M 34 -                                                                     27 F 27 -                                                                   ______________________________________                                        Mean Values ±                                                                   IDDM Patients  12 M/15 F                                                                              12.4 ± 6.8                                      (N = 27)                                                                      NIDDM Patients 3 M/2 F 57.0 ± 5.6                                          (N = 5)                                                                       Normal Subjects 18 M/10 F  35.8 ± 10.3                                   ______________________________________                                         All type 1 patients were treated with NPH insulin.                            The diagnosis of NIDDM diabetes in patients #26, and 27, the two oldest       patients was based on a history of ketoacidosis.                              Three of the NIDDM patients were receiving gliburide and were receiving       NPH insulin.                                                             

2. Glucose Uptake by Islet Cells

As shown in FIG. 1, the uptake of 3MG by islet cells, was consistentwith facilitated diffusion. The half time was less than 15 seconds andequilibrated with the extracellular 3MG concentration was more than 90%complete in 1 minute (FIG. 1). By virtue of the stereospecificity ofknown glucose transporters, L-glucose should be excluded from intactcells (14-16). In these studies L-glucose uptake at time zero was 19.2%of the urea space, increased slightly through 6 seconds and did notchange thereafter, thus providing evidence that the permeability barrierof the islet cells was intact (FIG. 1). L-glucose uptake was thereforedetermined at time zero in subsequent studies to provide an index of theextracellular space and cellular integrity.

3. Effects of IgG on 3MG Uptake into Islet Cells

An initial comparison of IgG from the first 8 participating IDDMpatients and 11 controls revealed significant inhibition of 3MGaccumulation at 3, 6, 16 and 30 seconds (p<0.01) but not at later timepoints. As would be expected in an incompletely inhibited facilitateddiffusion transport system, inhibition was greatest at the earliest timepoints. Subsequent comparisons in additional subjects were thereforerestricted to the 3, 6 and 15 second time points which provide the mostaccurate estimation of the initial rate of uptake. IgG from 27 new-onsetIDDM patients was found to inhibit significantly (p<0.01 at each timepoint) 3MG transport in dispersed rat islet cells compared to 28 controlsubjects (FIG. 2a). Progressive dilution of IgG resulted in progressiveloss of this inhibitory activity.

L-leucine uptake (FIG. 2b) was measured in tandem using IgG from 9 IDDMpatients and 15 control subjects to determine if the inhibition wasrestricted to 3MG transport or if inhibition of 3MG uptake represents anonspecific permeability alteration in islet cells exposed to islet cellantibodies. Uptake of L-leucine was virtually identical in islet cellsincubated in IDDM and control IgG.

The distribution of individual initial rates of 3MG uptake from alldeterminations using 28 nondiabetic, 27 IDDM and 5 NIDDM IgGpreparations is compared in FIG. 3. In islet cells incubated with IgGfrom patients with IDDM, initial rates of 3MG transport derived from the3, 6, and 15 second time points were 50% below control values (p<0.001).The rates in islet cells incubated with IgG from patients with NIDDM andother autoimmune diseases (w patients with Graves disease and 1 withsystemic lupus erythematosus) (not shown) were similar to the rates inthe presence of IgG from normal subjects. Reproducibility of the initialrate determinations were {1.8 mmoles 3MG min⁻¹ liter islet space⁻¹ from9 experiments using different IgG preparations from 4 IDDM patients.Data in FIG. 3 include only the first determination of each individualand do not include replicate experiments.

4. Effects of IgG on 3MG Concentration Dependence of 3MG Uptake

Examination of the glucose concentration dependence of3-0-methyl-β-D-glucose uptake by the islet cells used in this studyrevealed the presence of two kinetically distinct facilitated diffusiontransporters; one with an apparent Km of 18 mm and another with anapparent Km of 1.5 mM (J. H. Johnson, unpublished results). The Km 18 mMtransporter is kinetically similar to the liver transporter and studieswith an antibody to the liver transporter indicate that in the pancreasthe antibody is localized to β-cells (14,18). To test whether the IgGfractions from IDDM patients affects glucose transport by exerting theireffects preferentially on the Km 18 mM transporter or the Km 1.5 mMtransporter of β-cells, the glucose concentration dependance of3-0-methyl-β-D-glucose uptake was measured in islet cells incubated withIgG from 3 IDDM patients and 3 normal subjects. FIG. 4 shows that theIgG fractions from the IDDM patients inhibited β-cell Km 18 mM transportactivity without affecting Km 1.5 mM transport. The IgG fractions fromthe normal subjects did not alter these kinetics. Furthermore, the IgGfrom the IDDM patients decreased the maximum velocity of uptake withoutaltering the Km. These data further support an interaction between theIgG and β-cell glucose transporters.

5. Effects of Insulin and Insulin Antibodies upon 3-0-Methyl-β-D-GlucoseUptake by Normal Rat Islet Cells

The IgG fractions from the IDDM patients, all of whom had been treatedwith insulin for several weeks, may well have contained insulinantibodies (19) and/or autoantibodies (20) not present in the IgGfractions from the normal subjects. To exclude the possibility that suchantibodies might influence glucose uptake, 3-0-methyl-β-D-glucose uptakewas measured in the presence and absence of guinea pig anti-insulinserum. The guinea pig anti-insulin serum had no effect on islet cell3-0-methyl-β-D-glucose uptake rates being 9.8±0.8 mmoles/min/liter isletcell space in the presence of anti-insulin antibody (10 μg/ml, n=3)compared to 11.3±0.8 mmoles/min/liter islet space in the presence ofbuffer. The possibility that insulin copurified with insulin-bindingantibodies might bind to islets cells and somehow alter glucosetransport was also tested. The uptake rates of cells retreated for 20min. with both insulin and anti-insulin antibody took up3-0-methyl-β-D-glucose at a rate of 10.2±1.1 mmoles/min/liter isletspace. Therefore, neither insulin nor anti-insulin antibody nor thecombination affected islet cell 3-0-methyl-β-D-glucose uptake.

6. Effects of IgG on 3-0-Methyl-β-D-Glucose Uptake by Erythrocytes andHepatocytes

Recent evidence indicates that several structurally distinct facilitateddiffusion glucose transporters exist in mammalian tissues. The glucosetransporter from erythrocytes, adipocytes, and brain differs from thetransporter found in liver (21), whereas antibodies to a synthetic 13amino acids peptide from the predicted sequence of the C-terminal regionof the 55 kd rat liver glucose transporter reacts with a transporterexpressed in β-cells (18,21). The IgG fractions from 4 IDDM patients didnot inhibit 3-0-methyl-β-D-glucose uptake into human erythrocytes byalso failed to inhibit uptake by tat hepatocytes, despite the apparentsimilarity of liver and β-cell glucose transporters (FIG. 5).

7. Effects of Preincubation of IgG from IDDM patients with Islet-cellsand Liver, Erythrocyte and Kidney Plasma Membranes on Glucose TransportInhibition by IgG.

Adsorption experiments were performed to determine if the inhibitoryeffect of the 4IgG fractions from the IDDM patients on3-0-methyl-β-D-glucose transport in islet cells could be adsorbed bypreincubation with cells or cell membranes that do and do not expressthe glucose transporter shared by liver and β-cells. Four mg of IgG from4 IDDM patients was incubated with 20,000 islet cells, 20 mg hepatocytemembranes (cells that normally share the same glucose transporter), and20 mg of plasma membranes from rat erythrocytes and renal tubular cellbrush borders (neither of which contain this transporter). As shown inFIG. 6, the inhibitory effect was abolished by preincubation of the IgGwith islet cells and hepatocyte membranes but not by preincubation witherythrocyte or brush border membranes.

C. Discussion and Implications

The findings exemplified by studies such as the foregoing, areconsistent with the inventors' conclusion that autoimmune attacks onβ-cells include attacks on either a β-cell glucose transporter per se orsome other associated protein somehow involved in glucose transport. Thestudies show that IgGs obtained from the sera of humans with new-onsetIDDM inhibit initial rates of 3MG uptake into dispersed rat islets ofLangerhans. The inhibition of 3MG uptake by islets treated with theseIgGs does not appear to be a generalized reduction in islet cellpermeability since the uptake of L-leucine by these cells in unaffected.

The possibility of nonspecific toxic effects of β-cell antibodies onβ-cell viability as the explanation for these findings is excluded bythe fact that nonviable cells do not sediment through the dibutylphthalate layer of the assay tubes and are therefore not measured.Furthermore, the fact that L-glucose permeability was unaffected by thediabetic IgG indicates the integrity of the permeability barrier ofthese cell preparations.

Treatment of hepatocytes and erythrocytes with IgG from IDDM patientsdid not affect 3MG uptake into these cells. Thorens, et al. have shownthat the erythrocyte glucose transport protein has a 55% sequencehomology with the glucose transporter of liver indicating structuraldifferences between the two proteins (21). Antibodies to a syntheticpeptide deduced from the sequence of the liver transporter recognize aprotein of β-cells of islets, suggesting that these transporters may besimilar in structure (18). The lack of an effect of IgG from new-onsetIDDM patients upon glucose transport by hepatocytes was surprising andmay indicate that small structural differences between the β-celltransporter and the liver transporter do exist. Alternatively, IgG fromnew-onset IDDM patients might recognize an antigen other than thetransporter that is somehow functionally involved in 3MG uptake.

The results of the present invention suggest that islet cells have atransporter which is similar kinetically to the liver transporter, andthat IgG fractions from IDDM patients exert their inhibitory effectspreferentially on this high Km transporter. The fact that the inhibitoryeffect on 3-0-methyl-β-D-glucose uptake by islet cells was abolished bypreincubation with both islet cells and hepatocyte membranes, but not byerythrocyte or kidney tubule brush border membrane preparations,provides support for the interpretation that the liver and β-celltransporters are similar. The failure of IgG from IDDM patients toinhibit glucose transport by hepatocytes may indicate an excess ofglucose transporter relative to the concentration of putative antibody.

Antibodies to at least three different islet antigens have been found inserum of patients at the time of diagnosis of IDDM. These antigens arethe 64 kd membrane protein of Bedkkeskov et al.(5), cytoplasmic isletcell antigens (22), and insulin (20). However, there is no reason tobelieve that the 64 kd protein is related to the 55 kd glucosetransporter. Tests for cytoplasmic autoantibodies were negative inalmost half of the IDDM patients who IgG inhibited 3MG transportactivity. Although insulin autoantibodies were not assayed in the IDDMpatients, we established that preincubation of islet cells with insulinantibodies had no effect on 3-0-methyl-β-D-glucose uptake.

The functional implications of a 50% reduction in 3-0-methyl-β-D-glucoseuptake by islet cells are unclear. Glucose transport capacity farexceeds the capacity to phosphorylate glucose (5), suggesting that thisdegree of interference with glucose transport is probably not sufficientto affect insulin secretion in vivo. However, the IgG concentrationsused were 20% of those, on a protein-to-cell ratio basis, used byKanatsuna, et al.(4) to demonstrate IgG-induced inhibition of insulinsecretion by islets. Also it should be noted that 3-0-methyl-β-D-glucoseuptake differs kinetically from β-cells (12) and which do not expressthe livertype glucose transporter. This could well have minimized theinhibition. Nevertheless, the inventors consider it unlikely that theantibody-induced inhibition of in vitro glucose uptake is the cause ofthe loss of glucose-stimulated insulin secretion in vivo.

Even though there is no reason to assume that the putative glucosetransporter antibodies are cytotoxic to β-cells, it is conceivable thatthey initiate a chain of events leading to loss of glucose-stimulatedinsulin release. Alternatively they may be an epiphenomenon, an immuneresponse to release of β-cell antigens following destruction of β-cellsby an unidentified process.

REFERENCES

1. Lernmark et al. (1981), Diabetology, 21:431-35.

2. Ora et al. (1976), Pro. Natl. Acad. Sci. U.S.A., 73:1338-42.

3. Srikanta et al. (1983), N. Engl. Jrnl. Med., 308:322-25.

4. Kanatsuna et al. (1981), Diabetes, 30:231-34.

5. Baekeskov et al. (1982), Nature, 298:167-69.

6. Baekeskov et al. (1987), J. Clin. Invest., 79:926-34.

7. Christie et al. (1988), Diabetologia, 31:591-602.

8. Ey et al. (1978), Immunochem., 15:429-436.

9. Krell and Rabin (1984), Diabetes, 33: 703-11.

10. Naber et al. (1980), Diabetologia, 19:439-44.

11. Casten (1981), "Rotman and Papermasters Technic for FluorochromingViable Cells using FDA." In: Clark G, ed. Staining Procedures, 4th ed.Baltimore: Williams and Wilkin, pp. 93-94.

12. Gorus et al. (1984), J. Biol. Chem., 259:1196-1200.

13. Steck and Kant (1974), Meth. Enzymol., 31:172-180.

14. Axelrod and Pilch (1983), Biochemistry, 22:222-27.

15. Kaunitz and Wright (1984), J. Membr. Biol., 79:41-51.

16. Wheeler et al. (1981), J. Biol. Chem., 256:8907-14.

17. Hellman et al. (1971), Biochim. Biophys. Acta, 241:147-54.

18. Orci et al. (1989), Science, 245:295-297.

19. Berson et al. (1956), S. Clin. Invest., 35:170.

20. Palmer et al. (1983), Science, 223:1337-39.

21. Thorens et al. (1988), Cell, 55:281-90.

22. Botazzo et al. (1974), Lancet, 2:1297-83.

23. Ashcroft and Nino (1978), Biochim. Biophys. Acta., 538:334-42.

Modifications and alterations will become apparent to skilled artisansin light of the foregoing disclosure. It is intended by the inventorsthat all such modifications and changes be included within the scope ofthe subject matter which the present applicants regard as theirinvention, and as defined by the following claims.

What is claimed is:
 1. A method for diagnosing autoimmune insulindependent diabetes mellitus, comprising the steps of:(a) obtaining asample of serum from a patient suspected of having autoimmune IDDM; (b)testing for the presence of an autoimmune immunoglobulin in thepatient's serum the immunoglobulin being characterized by its ability tointerfere with the glucose transporting activity of pancreatic isletcell glucose transporter, the presence of such an immunoglobulin in thepatient's serum being diagnostic of autoimmune IDDM, wherein testing forthe presence of the immunoglobulin comprises:(i) preparing an admixturewhich includes the islet cell glucose transporter and serum from thepatient; (ii) incubating the admixture under conditions appropriate forthe formation of immunocomplexes; and (iii) testing for the formation ofimmunocomplexes between the islet cell glucose transporter and the serumimmunoglobulins, to determine the presence of the autoimmuneimmunoglobulin therein.
 2. The method of claim 1, wherein IgG isprepared from the patient's immunoglobulin, and the IgG is employed toprepare the admixture of step (a).